Of the numerous blood-stage malaria antigens that have been identified in both infected humans and in animal models of this disease, the clearest rationale for a vaccine candidate exists for the merozoite surface protein-1 (MSP-1). This protein is synthesized as a large precursor during schizogony and is processed after merozoite egress from the erythrocyte into a series of proteolytic fragments. Despite the studies available on the MSP-1 protein, we have little definitive information on its role or roles in the functioning of plasmodium species. Its presence on all species of plasmodia examined to date, its regions of conservation such as the cysteine-rich C-terminal portion of the molecule, its resistance to being disrupted by gene knock-out, and its complex and specific processing all support the biological importance of MSP-1 to the parasite. A number of functions have been proposed for MSP-1 protein including participation in the initial binding of the merozoite to the red cell, involvement in signaling the parasite that it is now within the red cell to promote growth or playing a role in the egress of merozoites from the erythrocyte. Our current hypothesis is that MSP-1 is a multi-domain protein composed of a number of modular regions with different functions and that many, if not all, of these functions are mediated via protein-protein interactions either with MSP-1 itself or other plasmodial or host molecules. We have chosen to utilize and our preliminary observations support the potential of the yeast two-hybrid system for examining protein-protein interactions of MSP-1. We will address the following specific aims in this proposal: (1) Define the intramolecular interactions of the MSP-1 protein. Preliminary experiments have demonstrated an interaction between two different regions of MSP-1. We propose to construct a detailed map of each of the interacting regions on the MSP-1 molecule and investigate the biological significance of this interaction. (2) Define the intermolecular interactions of the MSP-1 protein. Preliminary experiments have identified two previously undefined proteins that may interact with the N- terminal fragment of MSP-1. Experiments are proposed to investigate the nature of these molecules at the molecular level and characterize their interaction with MSP-1. Additional experiments are proposed to conduct similar two-hybrid studies for the other fragments of MSP-1 and to investigate additional relevant interactions through the construction of a bone marrow- derived cDNA library to explore potential parasite-host interactions. (3) Conduct a molecular analysis of the C-terminal 19 kDa fragment of MSP-1. Due to the cysteine-rich nature of this region, it has presented obstacles in using the two-hybrid approach. We propose alternative approaches to investigating the protein-protein interactions of this region. Finally, we describe a combinatorial chemistry approach to isolate small molecules that interfere with critical functions associated with this domain of MSP-1. We anticipate that this detailed analysis of the functioning of MSP-1 may lead to identification of new targets for immunologic or chemotherapeutic intervention, in addition to aiding our understanding of the biology of this key parasite molecule.